CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes

Ermert, Anna Lena; Nogué, Fabien; Stahl, Fabian; Gans, Tanja; Hughes, Jon

doi:10.1007/978-1-4939-9612-4_20

Cited by 10 publications

(15 citation statements)

References 37 publications

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“…Dual-cut experiments exploiting NHEJ and HDR (Tables 2 and 3 ) (Ermert et al 2019 ) are illustrated in Fig. 1 .…”

Section: Methodsmentioning

confidence: 99%

“…Sequences were aligned using Clustal and inspected for mutations around the PAM sequence. Potential mutants produced in dual-cut transfections were tested by PCR using primers flanking the two target sites as previously described (Ermert et al 2019).…”

Section: Knockout and Off-target Screeningmentioning

confidence: 99%

“…b Lanes 12-17 and 18-22 show the same extracts amplified with Taq DNA Polymerase. Lanes 14 & 16 and 19 & 22 show 2 & 1 bp deletions, respectively et al 2016) according to established procedures(Ermert et al 2019;Lopez-Obando et al 2016)…”

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confidence: 99%

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Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

et al. 2020

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Key message We mutated all seven Physcomitrium (Physcomitrella) patens phytochrome genes using highly-efficient CRISPR-Cas9 procedures. We thereby identified phy5a as the phytochrome primarily responsible for inhibiting gravitropism, proving the utility of the mutant library. Abstract The CRISPR-Cas9 system is a powerful tool for genome editing. Here we report highly-efficient multiplex CRISPR-Cas9 editing of the seven-member phytochrome gene family in the model bryophyte Physcomitrium (Physcomitrella) patens. Based on the co-delivery of an improved Cas9 plasmid with multiple sgRNA plasmids and an efficient screening procedure to identify high-order multiple mutants prior to sequencing, we demonstrate successful targeting of all seven PHY genes in a single transfection. We investigated further aspects of the CRISPR methodology in Physcomitrella, including the significance of spacing between paired sgRNA targets and the efficacy of NHEJ and HDR in repairing the chromosome when excising a complete locus. As proof-of-principle, we show that the septuple phy− mutant remains gravitropic in light, in line with expectations, and on the basis of data from lower order multiplex knockouts conclude that phy5a is the principal phytochrome responsible for inhibiting gravitropism in light. We expect, therefore, that this mutant collection will be valuable for further studies of phytochrome function and that the methods we describe will allow similar approaches to revealing specific functions in other gene families.

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“…Dual-cut experiments exploiting NHEJ and HDR (Tables 2 and 3 ) (Ermert et al 2019 ) are illustrated in Fig. 1 .…”

Section: Methodsmentioning

confidence: 99%

Section: Knockout and Off-target Screeningmentioning

confidence: 99%

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Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

et al. 2020

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“…Moreover, these ancestral moss PHY genes occupy a phylogenetically distinct clade from angiosperm PHYs (2), and PHY gene duplication and diversification occurred independently in the bryophytes and angiosperms (41). These data imply that the PHY genes of Physcomitrella may have unique functions, unlike those described for angiosperm PHYs (40)(41)(42). Null mutations in four moss PHY loci (phy1 to -4) were generated by homologous replacement, as described previously in Mittmann et al (2).…”

Section: Light-regulated Cuticle Development: Phytochrome (Phy) Mutantsmentioning

confidence: 99%

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves

Qiao

Bourgault

Mohammadi

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal thatphyB1 phyB2double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show thatphymutants of the mossPhyscomitrella patensexhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

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“…The Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system has brought a remarkable development in genome engineering efficiency in different organisms during the past few years (Doudna and Charpentier, 2014;Albadri et al, 2017;Ermert et al, 2019;Pu et al, 2019;Song et al, 2019). Briefly, the CRISPR-cas system employs a guide RNA (gRNA) and an endonuclease, mostly a single nuclease Cas9 (Makarova et al, 2011;Chylinski et al, 2014), as the two working elements for a site directed DNA cutting.…”

Section: Crispr-casmentioning

confidence: 99%

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

Cen

Lin

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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In recent years, there has been a noticeable increase in research interests on the Fusarium species, which includes prevalent plant pathogens and human pathogens, common microbial food contaminants and industrial microbes. Taken the advantage of gibberellin synthesis, Fusarium fujikuroi succeed in being a prevalent plant pathogen. At the meanwhile, F. fujikuroi was utilized for industrial production of gibberellins, a group of extensively applied phytohormone. F. fujikuroi has been known for its outstanding performance in gibberellin production for almost 100 years. Research activities relate to this species has lasted for a very long period. The slow development in biological investigation of F. fujikuroi is largely due to the lack of efficient research technologies and molecular tools. During the past decade, technologies to analyze the molecular basis of host-pathogen interactions and metabolic regulations have been developed rapidly, especially on the aspects of genetic manipulation. At the meanwhile, the industrial fermentation technologies kept sustained development. In this article, we reviewed the currently available research tools/methods for F. fujikuroi research, focusing on the topics about genetic engineering and gibberellin production.

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CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes

Cited by 10 publications

References 37 publications

Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

Multiplex CRISPR-Cas9 mutagenesis of the phytochrome gene family in Physcomitrium (Physcomitrella) patens

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

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